Animals and Experimental Design
Five sexually mature, western-range ewes were used during the anestrous season (June) in Colorado. Anestrous status was confirmed by the absence of ovarian activity at slaughter. Anterior pituitary glands were collected following anesthesia with sodium pentobarbital and exsanguina-tion. Tissues were removed and immediately placed in ice cold dissociation medium. All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee and complied with National Institutes of Health (NIH) guidelines.
Anterior pituitary cells were dissociated as described previously. Briefly, the anterior pituitary gland was separated from the neurohypophysis and 0.5-mm slices were prepared using a Stadie-Riggs hand microtome. Slices were washed five times in dissociation medium (0.8% NaCl. 0.59% Hepes, 0.18% glucose. 0.02% KCl pH 7.3) and incubated. with gentle shaking. for 90 min with 1 mg/ml col-lagenase, 1 mg/ml hyaluronidase, and 0.1% DNase at 37°C. After dissociation. cells were washed five times in dissociation medium and, finally, dispersed in Dulbeccos minimal essential medium (DMEM) containing 30% wether serum. Cell viability, as assessed by trypan-blue-exclusion, was >90% and yield was 1.2 ± 0.05 X 108 cells/gland. Cells were seeded at a concentration of 4 X 105 cell/well in 24-well plates for secretion studies and 3 X 106 cells in 7-cm plates for analysis of mRNA. Cells were incubated for 72 h at 37°C in the presence of 95% O2 and 5% CO2. Chemicals and DMEM were purchased from Sigma Chemical Company (St. Louis, MO).